The hormone gastrin binds to a gastrin/cholecystokinin (CCK)-B receptor with high affinity via its 5 carboxy-terminal amino acids. The CCK-B/gastrin receptor is a cytoplasmic membrane protein which is coupled via a G protein to intracellular signal transduction pathways that in turn control the expression of various genes.
Gastrin is a peptide hormone which occurs in two forms, tetratriacontagastrin (G34) and heptadecagastrin (G17), and is synthesized and secreted by specialized cells, G cells, that are located in the stomach antrum. The hormone is secreted into the circulating blood and binds to specific cells in the stomach, namely, enterochromaffin-like (ECL) and parietal cells, that indirectly or directly affect stomach acid output. Historically, gastrin hormones have been associated with the stimulation of gastric acid secretion (Edkins, J. S. 1905). (The full citations for the references cited herein are provided in the Reference section preceding the claims.) In recent years, evidence has accumulated that gastrin may act as a trophic factor within the gastrointestinal tract (Johnson, L. 1997) and that it can promote the growth of gastrointestinal cancers (Watson et al. 1989, Dickinson, C. J. 1995), as well as non-gastrointestinal cancers including small cell carcinoma of the lung (Rehfeld et al. 1989). In the post-translational processing of gastrin, it is the xe2x80x9cmaturexe2x80x9d carboxy-amidated form that binds to the gastrin/CCK-B receptor via the carboxy terminus (Kopin et al. 1992).
It has been shown that several types of tumors, e.g., colorectal, stomach, pancreatic and hepatocellular adenocarcinomas possess CCK-B/gastrin receptors in their plasma membranes and that they respond to gastrin with powerful cellular proliferation (Rehfeld, J. F. 1972, Upp et al. 1989 and Watson et al. 1993). Furthermore, more recently it has been discovered that many of these cancer cells also secrete gastrin and thus effect an autonomous proliferative pathway (Van-Solinge et al. 1993, Nemeth et al. 1993 and Seva et al. 1994).
The CCK-B/gastrin receptor belongs to a family of G protein-coupled receptors with seven transmembrane domains with equal affinity for both CCK and gastrin (Soll et al. 1984). This receptor was named a CCK type-B receptor because it was found predominantly in the brain (Wank et al. 1992). The receptor was subsequently found to be identical to the peripheral CCK/gastrin receptor in the parietal and ECL cells of the stomach (Nakata et al. 1992). This receptor has been well characterized in a number of normal (Fourmy et al. 1984, Grider et al. 1990) and tumor tissues (Singh et al. 1990, Watson et al. 1993), and extensively studied using the rat pancreatic adenocarcinoma cell line AR42J (Scemama et al. 1987). The AR42J CCK-B/gastrin receptor cDNA has been cloned and sequenced, and it is more than 90% homologous in DNA sequence to the CCK-B/gastrin receptor in rat and human brain, and more than 84% homologous in sequence to the canine parietal cell CCK-B/gastrin receptor cDNA (Wank, S. A. 1995), demonstrating a high sequence homology even between species.
The peptide hormones G17 and G34 bind to the CCK-B/gastrin receptor on the cell membrane of normal cells. However, it has been found that G17, and not G34, stimulates the growth of gastrin-dependent cancer cells. Serum-associated G17, in particular, has the potential to stimulate the growth of colorectal tumors in an endocrine manner mediated by CCK-B/gastrin receptors (Watson et al. 1993) in the tumor cells. Gastrin-17 appears to be particularly implicated in stimulating the growth of colorectal adenocarcinomas due to a possible increased affinity for the CCK-B/gastrin receptor on the tumor cells, over other gastrin hormone species (Rehfeld 1972 and 1993). The CCK-B/gastrin receptors were found to be expressed in a high affinity form on 56.7% of human primary colorectal tumors (Upp et al. 1989). It has been postulated that a potential autocrine loop may also exist due to endogenous production of precursor gastrin peptides by such tumors (Van-Solinge et al. 1993 and Nemeth et al. 1993). The resulting G17 ligand/receptor complex stimulates cell growth by way of secondary messengers for regulating cell function (Ulirich et al. 1990). The binding of G17 to the CCK-B/gastrin receptor leads to activation of phosphatidyl inositol breakdown, protein kinase C activation with a resultant increase in intracellular calcium ion concentration, as well as the induction of c-fos and c-jun genes via mitogen-activated protein kinase, which has been implicated in the regulation of cell proliferation (Tadisco et al. 1995). Additionally, gastrin binding to the CCK-B/gastrin receptor has been associated with the subsequent increase in phosphorylation by a tyrosine kinase, pp125FADK (focal adhesion kinase), which may also have a role in the transmission of mitogenic signals (Tanaguchi et al. 1994).
A number of high affinity CCK-B/gastrin receptor antagonists have been evaluated therapeutically both in vitro and in vivo in a number of experimental gastrointestinal cancers. For example, proglumide, a glutamic acid derivative (Seva et al. 190; Harrison et al. 1990 and Watson et al. 1991a); Benzotript, an N-acyl derivative of tryptophan; L-365,260, a derivative of Aspercillin (Bock et al. 1989), and CI-988 a molecule that mimics the C-terminal pentapeptide sequence of CCK (Hughes et al. 1990) have been shown to effectively neutralize the effects of exogenous gastrin on gastrointestinal tumor growth both in vitro and in vivo (Watson et al. and Romani et al. 1994). However, these antagonists have severe toxic side effects and lack specificity as they block the action of all potential ligands of the receptor such as G34 and CCK in normal cells. Recently, highly potent and selective CCKB/gastrin receptor antagonists such as YM022 (Yuki et al., 1997) and YF476 (Takinami et al., 1997) have been also described.
Proglumide and Benzotript have been widely assessed in the pre-clinical studies. The main problem with these compounds is their lack of potency, with relatively high concentrations required to displace G17 (Watson et al., 1992a; Watson et al., 1992b). Despite this, proglumide and benzotript inhibited the basal and gastrin-stimulated proliferation of a number of cell lines (Seva et al., 1990; Watson et al., 1991a). In addition, proglumide increased the survival of xenograft mice bearing the gastrin-sensitive mouse colon tumor, MC26 to 39 days in the treated animals from 25 days in the control animals.
Due to the low specificity of this class of gastrin antagonising agents for the gastrin/CCKB receptor, the inhibition of tumor growth may not be effectively control with gastrin antagonists. Moreover, the cellular receptors which recognize and bind the gastrins do not bind all the inhibitors tested (Seva et al. 1994). Thus, if complete inhibition of gastrin binding to the receptor does not occur in the autocrine growth cascade, then the gastrin antagonists may be unable to block this mechanism of tumor growth promotion.
The present invention provides immunogenic compositions and immunological methods for the treatment of gastrin-dependent tumors. The method comprises the active or passive immunization of a patient with an anti-CCK-B/gastrin receptor immunogen or anti-CCK-B/gastrin receptor antibodies. The antibodies produced by the immunogens are specific against the CCK-B/gastrin receptor on tumor cells and block the growth-promoting effects of gastrin on the receptors. The antibodies prevent the peptide hormones from binding to the CCK-B/gastrin receptors on gastrin-dependent tumor cells; thus, the growth of the tumor is arrested. Furthermore, surprisingly, the antibodies specific to the NH2-terminal end of the receptor, upon binding to the receptor, are internalized and rapidly translocated into the cytoplasm and into the nucleus of the tumor cells. This internalization can occur as early as 10 seconds after exposing the cells to the antibody. This rapid internalization of the antibody/receptor complex in turn causes the affected tumor cells to undergo apoptosis or suicide.
The immunogens of the invention comprise natural or synthetic peptides derived from the human CCK-B/gastrin receptor, as the immunomimic portion of the immunogen. The immunogens may also comprise a spacer peptide sequence attached to an end of the immunomimic peptide. The immunogen may also be conjugated to a protein carrier, such as Diphtheria toxoid, tetanus toxoid, bovine serum albumin and the like.
In one embodiment, the method of immunization against the CCK-B/gastrin receptor comprises active immunization, wherein a patient is immunized with an immunogen of the invention. The immunogen stimulates the production of antibodies against the CCK-B/gastrin receptor on tumor cells.
The antibodies produced by the anti-CCK-B/gastrin receptor immunogens bind to the CCK-B/gastrin receptors on tumor cells and effectively prevent the binding of the peptide hormones to the receptors, thereby inhibiting the autocrine growth-stimulatory pathway of tumor cell division and ultimately the growth of the tumor.
In another embodiment of the invention, the method of treatment comprises passive immunization, whereby antibodies against the CCK-B/gastrin receptor are administered to a patient in a sufficient concentration to bind to the CCK-B/gastrin receptors of the tumor cells, and the antibodies block the binding of the peptide hormones to the receptor. The prevention of binding of the hormones to their receptor inhibits the growth-stimulus pathway of the tumor cells, thereby inhibiting the growth of the hormone-dependent tumors. In a preferred embodiment of this aspect of the invention, the antibodies for human therapy may be chimeric, humanized, or human monoclonal antibodies which may be produced by methods well known in the art. In addition, the anti-CCK-B/gastrin receptor antibodies may be further conjugated to cytotoxic molecules such as cholera toxin, or to radioactive molecules labeled with a radionuclide, such as 125I and 131I, to enhance the killing of the tumor cells.
The invention also provides a method for diagnosing a gastrin-responsive tumor, comprising the immunochemical detection of gastrin-dependent (CCK-B/gastrin-containing) tumors from a tissue biopsy using the antibodies of the invention. The specific anti-CCK-B/gastrin receptor antibodies of the invention can be labeled with a detection system utilizing compounds such as biotin, horseradish peroxidase and fluorescein to detect the CCK-B/gastrin receptors in the tumor tissue using standard immunochemical procedures.
The invention also provides a method for diagnosing a gastrin-dependent tumor, comprising the in vivo detection of gastrin-dependent (CCK-B/gastrin receptor-containing) tumors, using the anti-CCK-B/gastrin receptor antibodies. The method comprises, administering to a patient possessing a colorectal tumor an effective dose of radiolabeled anti-CCK-B/gastrin receptor antibodies via an intravenous injection, and imaging or detecting tumor cells having anti-CCK-B/gastrin receptor antibodies bound to their cell membranes by standard scintigraphic scanning procedures. In this aspect of the invention, the anti-CCK-B/gastrin-receptor-antibodies should be labeled with a radionuclide such as 111Indium, 90Yttrium, and 131I.